This invention relates to materials and methods for modulating the activity of HIV RNA. The invention generally relates to the field of "antisense" compounds, compounds which are capable of specific hybridization with a nucleotide sequence of an RNA. In accordance with preferred embodiments, this invention is directed to methods for achieving therapeutic treatment of disease and regulating gene activity.
It is well known that most of the bodily states in mammals including infectious disease states, are effected by proteins. Such proteins, either acting directly or through their enzymatic functions, contribute in major proportion to many diseases in animals and man. Classical therapeutics has generally focused upon interactions with such proteins in efforts to moderate their disease causing or disease potentiating functions. Recently, however, attempts have been made to moderate the actual production of such proteins by interactions with molecules that direct their synthesis, intracellular RNA. By interfering with the production of proteins, it has been hoped to effect therapeutic results with maximum effect and minimal side effects. It is the general object of such therapeutic approaches to interfere with or otherwise modulate gene expression leading to undesired protein formation.
One method for inhibiting specific gene expression which has been adopted to some degree is the "antisense" approach, where oligonucleotide analogs complementary to a specific, target, messenger RNA, MRNA sequence are used. A number of workers have reported such attempts. Pertinent reviews include C. A. Stein & J. S. Cohen, Cancer Research, vol. 48, pp. 2659-2668 (1988); J. Walder, Genes & Development, vol. 2, pp. 502-504 (1988); C. J. Marcus-Sekura, Anal. Biochemistry, vol. 172, 289-295 (1988); G. Zon, Journal of Protein Chemistry, vol. 6, pp-131-145 (1987); G. Zon, Pharmaceutical Research, vol. 5, pp. 539-549 (1988); A. R. Van der Krol, J. N. Mol, & A. R. Stuitje, BioTechniques, vol. 6, pp. 958-973 (1988) and D. S. Loose-Mitchell, TIPS, vol. 9, pp. 45-47 (1988). Each of the foregoing provide background concerning general antisense theory and prior techniques.
Prior attempts to inhibit HIV by various antisense approaches have been made by a number of researchers. Zamecnik and coworkers have used phosphodiester oligonucleotides targeted to the reverse transcriptase primer site and to splice donor/acceptor sites, P. C. Zamecnik, J. Goodchild, Y. Taguchi, P. S. Sarin, Proc. Natl. Acad. Sci. USA 83, 4143 (1986). Goodchild and coworkers have made phosphodiester compounds targeted to the initiation sites for translation, the cap site, the polyadenylation signal, the 5' repeat region and a site between the gag and pol genes. J. Goodchild, S. Agrawal, M. P. Civeira, P. S. Sarin, D. Sun, P. C. Zamecnik, Proc. Natl. Acad. Sci. U.S.A. 85, 5507 (1988). In the Goodchild study, the greatest activity was achieved by targeting the polyadenylation signal. Agrawal and coworkers have extended the studies of Goodchild by using chemically modified oligonucleotide analogs which were also targeted to the cap and splice donor/acceptor sites. S. Agrawal, J. Goodchild, M. P. Civeira, A. H. Thornton, P. S. Sarin, P. C. Zamecnik, Proc. Nat'l. Acad. Sci. USA 85, 7079 (1988). A portion of one of these overlapped a portion of the HIV TAR region but was not found to have exemplary effect. Neither was this oligonucleotide analog designed to interfere with the HIV TAR region. Agrawal and coworkers have used oligonucleotide analogs targeted to the splice donor/acceptor site to inhibit HIV infection in early infected and chronically infected cells. S. Agrawal, T. Ikeuchi, D. Sun, P. S. Sarin, A. Konopka, J. Maizel, Proc. Natl. Acad. Sci. U.S.A. 86, 7790 (1989).
Stropp et al. (European Patent Application No. 90103541.8) disclose chemically modified antisense oligonucleotides for inhibiting TAR and the synthesis of the tat protein from HIV-1 and the use of these oligonucleotides in drugs for the treatment of HIV infections. The oligonucleotides and oligonucleosides are derivatives of normal deoxyribonucleic acid oligomers substituted especially at the phosphorus diester bonds and also at the 3' and 5' termini so that they are resistant to nuclease decomposition and have better permeation or hybridization properties. Stropp et al. found that the antisense oligonucleotides having specific sequences showed significantly greater inhibitory activity than prior art oligonucleotides. It was also found that normal deoxyribonucleic acid oligonucleotides were almost completely decomposed in cell culture tests in 2-3 hours. The chemically modified oligonucleotides used by Stropp et al. (phosphorothioates and methyl phosphonates) showed high nuclease resistance, as compared to the oligonucleotides of Goodchild et al. (1988). Stropp et al. disclose antisense oligonucleotides against the following TAR and leader sequences: nucleotides 21-53, 74-161, 202-279 and the second and third exon of the tat gene, nucleotides 5368-5403, 5421-5548, 5583-5617, and nucleotides 7967-8366, 8385-9183 of the HIV-1 genome. In the tests performed, it was found that these oligonucleotides inhibited protein synthesis in the 5' untranslated region, the 3' end of the tat mRNA, and within the tat mRNA. Phosphorothioate oligonucleotides which hybridize in the loop region of TAR MRNA were found to be very effective. Comparative studies with respect to the number of nucleotide monomers showed that effective translation inhibition was achieved with 10-18-mer oligonucleotides. Replacement of one or more bases with other partial structures (e.g., hypoxanthine or other purines, pyrimidines) did not impair hybridization.
Shibahara et al. (European Patent Application No. 89303700.2) disclose 2'-0-methylribo oligonucleotide analogs, phosphorothioate 2-O-methylribo oligonucleotide analogs, and oligonucleotide derivatives complementary to the HIV virus mRNA splice site, to the primer-tRNA binding site, to the region upstream from the gag gene initiation codon, to a region internal to the gag gene, and to nucleotides 19-38 of the TAR element.
Sarin and coworkers have also used chemically modified oligonucleotide analogs targeted to the cap and splice donor/acceptor sites. P. S. Sarin, S. Agrawal, M. P. Civeira, J. Goodchild, T. Ikeuchi, P. C. Zamecnik, Proc. Natl. Acad. Sci. U.S.A. 85, 7448 (1988). Zaia and coworkers have also used an oligonucleotide analog targeted to a splice acceptor site to inhibit HIV. J. A. Zaia, J. J. Rossi, G. J. Murakawa, P. A. Spallone, D. A. Stephens, B. E. Kaplan, J. Virol. 62, 3914 (1988). Matsukura and coworkers have synthesized oligonucleotide analogs targeted to the initiation of translation of the rev gene mRNA. M. Matsukura, K. Shinozuka, G. Zon, et al, Proc. Natl. Acad. Sci. USA 84, 7706 (1987); R. L. Letsinger, G. R. Zhang, D. K. Sun, T. Ikeuchi, P. S. Sarin, Proc. Natl. Acad. Sci. U.S.A. 86, 6553 (1989). Mori and coworkers have used a different oligonucleotide analog targeted to the same region as Matsukura. K. Mori, C. Boiziau, C. Cazenave, et al., Nucleic Acids Res. 17, 8207 (1989). Shibahara and coworkers have used oligonucleotide analogs targeted to a splice acceptor site as well as the reverse transcriptase primer binding site. S. Shibahara, S. Mukai, H. Morisawa, H. Nakashima, S. Kobayashi, N. Yamamoto, Nucl. Acids Res. 17, 239 (1989). Letsinger and coworkers have synthesized and tested oligonucleotide analogs with conjugated cholesterol targeted to a splice site. K. Mori, C. Boiziau, C. Cazenave, et al., Nucleic Acids Res. 17, 8207 (1989). Stevenson and Iversen have conjugated polylysine to oligonucleotide analogs targeted to the splice donor and the 5'-end of the first exon of the tat gene. M. Stevenson, P.L. Iversen, J. Gen. Virol. 70, 2673 (1989).
These prior attempts at targeting HIV have largely focused on the nature of the chemical modification used in the oligonucleotide. Although each of the above publications have reported some degree of success in inhibiting some function of the virus, a general therapeutic scheme to target HIV and other retroviruses has not been found. Accordingly, there has been and continues to be a long-felt need for the design of oligonucleotides which are capable of effective, therapeutic antisense use.
This long-felt need has not been satisfied by prior work in the field of antisense oligonucleotide therapy for HIV and other retroviruses and viruses. Others have failed to identify target sites in which antisense oligonucleotides s are therapeutically effective at reasonable rates of application.